Proteolytic enzymes may play a role in the pathophysiology of degenerative diseases of the brain. The multicatalytic proteinase complex (MPC) first isolated and characterized in our laboratory from bovine pituitaries a decade ago, is a unique high molecular mass proteinase (700 kDa), composed of multiple non-identical subunits, and containing at least three distinct proteolytic activities. The primary structures of its cloned subunits which are highly conserved, bear no homology to any known protein but are homologous to each other. MPC is present in relatively high concentrations in brain and pituitary cytoplasm, where it likely participates in the extralysosomal degradation of ubiquitinated and non-ubiquitinated proteins. Many questions about the mechanism of its regulation and the relationship of its structure to its properties remain unanswered. We have found that chemical modification is a powerful approach to the study of structure- function relationships. Acetylation with N-acetylimidazole revealed that the initial degradation of casein may be catalyzed by a fourth component, distinct from the three components previously described and demonstrated that it is possible to prepare different catalytically active forms of the enzyme. The studies proposed in this application will employ chemical modification to characterize the individual components. N-acetylimidazole, citraconic anhydride and succinic anhydride will be used to determine the relationship of quaternary structure to function, and to prepare forms of the enzyme for characterization of the substrate specificities of each of the components. Active-site directed inhibitors will be synthesized. MPC has been isolated in both latent and activated forms. The mechanisms underlying its activation remain unknown. We have demonstrated that several MPC subunits are phosphorylated by a cAMP-dependent protein kinase that co-purifies with the proteinase. Phenyl Sepharose chromatography removes the kinase as well as the heaviest MPC subunit, while activating the degradation of casein. The possible role of phosphorylation and of the heaviest MPC subunit in the regulation of MPC activity will be explored by the application of classical and molecular biological techniques. It is likely that MPC abnormalities underlie some brain pathologies. The proposed studies should aid in our understanding of this biologically significant macromolecule.